Brain, Vol. 123, No. 3, 591-600,
March 2000
© 2000 Oxford University Press
Invited review |
Cytochrome oxidase immunohistochemistry: clues for genetic mechanisms
1 Metabolic Unit, Institute of Child Health, University College, 2 University Department of Clinical Neurosciences, Royal Free Hospital and 3 University Department of Clinical Neurology, Institute of Neurology, London
Correspondence to:
Dr Shamima Rahman, Metabolic Unit, Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK E-mail: S.Rahman{at}ich.ucl.ac.uk
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Abstract |
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Cytochrome c oxidase (COX) is encoded by three mitochondrial and nine nuclear genes. COX deficiency is genetically heterogeneous but current diagnostic methods cannot easily distinguish between mitochondrial and nuclear defects. We hypothesized that there may be differential expression of COX subunits depending on the underlying mutation. COX subunit expression was investigated in five patients with known mtDNA mutations. Severe and selective reduction of mtDNA-encoded COX subunits I and II was consistently observed in all these patients and was restricted to COX-deficient fibres. Immunostaining of nuclear-encoded subunits COX IV and Va was normal, whilst subunit VIc, also nuclear-encoded, was decreased. Twelve of 36 additional patients with histochemically defined COX deficiency also had this pattern of staining, suggesting that they had mtDNA defects. Clinical features in this group were heterogeneous, including infantile encephalopathy, multisystem disease, cardiomyopathy and childhood-onset isolated myopathy. The remaining patients did not have the same pattern of immunostaining. Fourteen had reduced staining of all subunits, whilst 10 had normal staining of all subunits despite reduced enzyme activity. Patients with COX deficiency secondary to mtDNA mutations have a specific pattern of subunit loss, but the majority of children with COX deficiency do not have this pattern of subunit loss and are likely to have nuclear gene defects.
cytochrome oxidase; immunohistochemistry; mitochondria; mtDNA
COX = cytochrome c oxidase; DAB = 3,3'-diaminobenzidine hydrochloride; MELAS = mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes; MERRF = myoclonus epilepsy and ragged red fibres; mtDNA = mitochondrial DNA; PDHC = pyruvate dehydrogenase complex; PBS = phosphate-buffered saline; SDH = succinate dehydrogenase; tRNA = transfer RNA
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Introduction |
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Cytochrome c oxidase (COX), or complex IV of the mitochondrial respiratory chain, catalyses the transfer of electrons from reduced cytochrome c to molecular oxygen, and couples this reaction to proton pumping across the inner mitochondrial membrane. The human enzyme is composed of 13 polypeptide subunits that are of dual genetic origin. The three major subunits (I, II and III) constitute the catalytic core of the enzyme (Capaldi, 1990
) and are encoded by mitochondrial DNA (mtDNA). The mitochondrial genome is a maternally inherited circular 16 569 bp (base pair) double-stranded DNA molecule encoding 22 transfer RNAs (tRNAs), two ribosomal RNAs and 10 other respiratory chain enzyme subunits in addition to the three subunits of COX (Anderson et al., 1981). The remaining subunits of COX (IV, Va, Vb, VIa, VIb, VIc, VIIa, VIIb, VIIc and VIII) are nuclear-encoded, synthesized on cytosolic ribosomes and subsequently transported into the mitochondria. It is postulated that the nuclear subunits may be involved in the assembly or stability of the holoenzyme complex, and/or may have regulatory functions by binding ligands that modulate the catalytic function of the enzyme.
COX deficiency, either total or partial, is the most commonly recognized respiratory chain defect in childhood (Caruso et al., 1996). This may be an isolated defect or may be combined with deficiencies of other components of the respiratory chain. The availability of a reliable cytochemical method to visualize COX activity (Seligman et al., 1968) means that COX deficiency can be detected in very small muscle biopsies, even when there is insufficient tissue for measurement of enzyme activity. Clinical presentations are very heterogeneous and include fatal infantile myopathy with or without a Fanconi-type renal tubulopathy (DiMauro et al., 1980), Leigh syndrome (subacute necrotizing encephalomyelopathy) (Willems et al., 1977; Rahman et al., 1996), cardiomyopathy and myopathy (Zeviani et al., 1986), recurrent myoglobinuria (Saunier et al., 1995; Keightley et al., 1996) and a `benign' spontaneously reversible COX-deficient myopathy (DiMauro et al., 1983). In addition, partial COX deficiency may be seen in certain well-recognized mitochondrial syndromes, such as the Kearns–Sayre, MELAS (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes) and MERRF (myoclonus epilepsy and ragged red fibres) syndromes (Johnson et al., 1983; Hammans et al., 1995; Taanman, 1997). These syndromes are associated with mtDNA mutations, and the COX deficiency is patchy with some fibres deficient and others apparently normal, reflecting the heteroplasmy of the mutations. A critical mutant load is required to produce a biochemical defect, and fibres with mutant loads below this threshold appear histochemically normal (Schon et al., 1997).
Mutations have been identified in all three mtDNA-encoded COX subunit genes. Most reported mutations are in the COX subunit III gene: a 15 bp microdeletion in a highly conserved region of the gene was associated with recurrent myoglobinuria and myopathy in a 15-year-old girl (Keightley et al., 1996); a missense mutation at position 9957 was found in an adult with MELAS (Manfredi et al., 1995); and a stop mutation at nucleotide 9952 was described in a 36-year-old woman suffering from episodes of encephalopathy associated with lactic acidaemia, exercise intolerance and proximal myopathy (Hanna et al., 1998b). A heteroplasmic 5 bp microdeletion in the mitochondrial COX subunit I gene has been described in association with severe isolated muscle COX deficiency in a patient with atypical motor neuron disease (Comi et al., 1998). Recently, a point mutation in the initiation codon of the COX subunit II gene was reported in a patient with encephalomyopathy (Clark et al., 1999).
As most subunits of COX are nuclear-encoded and additional nuclear-encoded factors are essential for assembly of the holoenzyme complex, it is likely that most cases of COX deficiency are caused by nuclear gene mutations. However, mutations have yet to be described in the nuclear COX subunit genes, despite extensive sequence analysis (Adams et al., 1997; Lee et al., 1998). Characterization of human nuclear-encoded assembly and import factors and other nuclear regulatory genes for COX is still in its infancy. Recently, mutations have been identified in the SURF1 gene on chromosome 9 in patients with Leigh syndrome and COX deficiency (Tiranti et al., 1998; Zhu et al., 1998). The SURF1 gene product is thought to be involved in COX assembly or maintenance.
Thus, most patients with COX deficiency remain uncharacterized at the molecular level. In the absence of an identified mtDNA mutation or a strong maternal family history of neuromuscular disease it is difficult to be certain whether the genetic defect is mitochondrial or nuclear in individual cases. This leads to difficulties in genetic counselling. We sought to determine whether COX subunit expression patterns detected immunohistochemically, using highly specific monoclonal antibodies, can distinguish between mtDNA defects and nuclear DNA defects in COX deficiency. This would be an invaluable aid to genetic counselling and would help to direct subsequent molecular genetic investigations. In addition, studying patterns of subunit expression in COX-deficient patients is fundamental to understanding the pathogenesis of respiratory chain enzyme deficiencies.
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Patients |
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Muscle biopsies with decreased or absent COX activity, as determined by histochemistry, were identified retrospectively from the histopathological records at Great Ormond Street Hospital for Children. These muscle biopsies had been obtained, after informed parental consent, for diagnostic purposes from children with suspected respiratory chain disorders over a 12-year period (1985–97). Patients were excluded from the study only if no frozen muscle remained. We studied 36 children with reduced COX staining in biopsied skeletal muscle in whom the molecular defect was uncertain. Clinical features are summarized in Table 1
(P1 to P36). Patients were aged between 5 days and 15 years 10 months (median 1 year 5 months) at the time of biopsy, the majority being infants. There was a male preponderance of 1.8 : 1. Lactate levels were raised in the blood and/or CSF in 34 of 35 patients. Lactate : pyruvate ratios were determined in 19 patients and were elevated in 16 of these. Nine patients had Leigh syndrome, defined by clinical features, lactic acidosis and neuroimaging.
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We also examined muscle from five patients with known mtDNA defects (C1 to C5 in Table 1
). Three had point mutations involving tRNA genes: insertion of a cytosine at nucleotide position 7472 in patient C1 (Hanna et al., 1998a); T14709C in patient C2 (Hanna et al., 1995); and A8344G in patient C5 (Sweeney et al., 1994). Patient C4 had a point mutation in the COX subunit III gene (G9952A) (Hanna et al., 1998b) whilst patient C3 had the `common' 4.9 kb mtDNA deletion in association with clinical features of the Pearson and Kearns–Sayre syndromes (McShane et al., 1991). Three normal control biopsies had no histological evidence of muscle disease. Three other biopsies with normal quantitative COX activity were obtained from patients with congenital lactic acidosis associated with isolated deficiencies of pyruvate carboxylase, the pyruvate dehydrogenase complex (PDHC) and complex I of the respiratory chain.
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Material and methods |
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Open or needle muscle biopsies were performed under general anaesthesia and snap frozen immediately in hexane maintained in an acetone/CO2 bath at –80°C. After sectioning for diagnostic purposes, they were stored at –50°C until the time of this study.
Histochemistry
For histochemical studies, cryostat sections cut at 10 µm were stained to demonstrate the activities of succinate dehydrogenase (SDH) and COX using standard methods (Filipe and Lake, 1990; Stoward and Pearse, 1991). Sections were also stained with the modified Gomori trichrome, and for glycogen and fat. Muscle fibres were typed using standard ATPase techniques, with and without acid preincubation.
Antibodies
A battery of subunit-specific monoclonal antibodies was used to identify COX subunits immunohistochemically. Mouse monoclonal antibodies directed against subunits I and II (mitochondrial-encoded) and IV, Va and VIc (nuclear-encoded) were used (Taanman et al., 1996). A mouse monoclonal antibody directed against fast myosin (Novocastra, Newcastle-upon-Tyne, UK) was used to identify type I and type II muscle fibres.
Immunohistochemistry
Serial 8-µm cryostat sections were cut from patient and control biopsies, mounted adjacently on polysine slides (BDH, Poole, UK) and air-dried for 1 h. Mouse monoclonal antibodies directed against COX subunits and fast myosin were optimally diluted in phosphate-buffered saline (PBS), pH 7.4, and 200 µl of each was applied to serial sections for each patient and control. Sections were incubated with primary antibody overnight in a humidified chamber to prevent dehydration. One set of sections was incubated in PBS alone, without primary antibody, as a control. All sections were washed in PBS and then incubated for 45 min in biotinylated rabbit anti-mouse IgG secondary antibody (Dako, Glostrup, Denmark). The immunoreaction was visualized after a 45 min incubation in streptavidin–biotinylated horseradish peroxidase complex (ABC complex, Dako) by developing the peroxidase activity in a solution containing 0.05% 3,3'-diaminobenzidine hydrochloride (DAB) and 0.03% hydrogen peroxide in PBS for 10 min. Sections were washed with PBS between each step of the procedure, and finally in tap water for 10 min after incubating in DAB. Carazzi's haematoxylin was used as a nuclear counterstain. Sections were dehydrated, cleared and mounted in DPX. All incubations were at room temperature.
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Results |
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Histochemistry
All patients had reduced COX activity on histochemical staining. In some cases there was complete absence of COX staining, whilst in others there was a more patchy loss, with variation in staining intensity unrelated to fibre type (Table 2
). In 15 patients, the decreased COX staining activity shown histochemically was associated with COX enzyme activity below the normal range on biochemical assay (Table 1). In four patients, COX activity was at the bottom end of the normal range. In three of these there were some COX-negative fibres on histochemistry, whilst the fourth had weaker histochemical staining for COX than a control biopsy stained alongside on the same slide. There was insufficient material for performing enzyme assays in the other patients. All but two patients (P14 and P20) had increased neutral lipid droplets within their muscle fibres. Only three cases (P1, P9 and P15) had classical ragged red fibres on Gomori trichrome staining, but 20 had evidence of mitochondrial proliferation suggested by increased SDH staining.
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Immunohistochemistry
Clear particulate immunoreactivity consistent with mitochondrial staining was observed in muscle fibres from normal control biopsies with all antibodies directed against COX subunits. Permeabilization, as used by Johnson and colleagues (Johnson et al., 1988
), had no effect on the staining intensity in control samples and was not used in this study. Immunoreactivity was more prominent in type I fibres, which have a higher mitochondrial content. No immunostaining was observed in sections incubated with PBS, pH 7.4, in place of primary antibody, as a negative control. Patients with proven pyruvate carboxylase, PDHC and isolated complex I deficiency also had normal immunostaining of all COX subunits as well as normal COX activity.
mtDNA mutations
All five patients with known mtDNA mutations had an identical pattern of COX subunit immunostaining, with selective loss of staining of mitochondrial subunits I and II, together with reduced staining of subunit VIc (Fig. 1). This reduction was limited to COX-deficient fibres. All subunits stained normally in COX-positive fibres. The other 36 patients fell into two groups, defined by their immunostaining. In those biopsies in which fibres were COX-positive by histochemistry, these same fibres stained positively with the range of COX monoclonal antibodies used. For COX-negative fibres, however, the pattern of immunostaining varied according to the classification outlined.
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Group 1 (Table 2
)Ten of the COX-deficient patients with unknown aetiology had a pattern of COX immunostaining similar or identical to that of the five patients with mtDNA mutations (Fig. 1
). One of these patients (P26) had reduction of subunit II only, with normal staining of subunit I. Subunit VIc was also reduced in six of these 10 patients, including the patient with isolated reduced staining of subunit II. This group of patients was clinically heterogeneous, including patients with encephalopathy in infancy, multisystem disease, cardiomyopathy and isolated myopathy. Screening for mtDNA deletions, point mutations at nucleotides 3243, 8344 and 8993 and for mtDNA depletion was negative in these 10 patients. Two other patients, with mtDNA depletion on Southern blot analysis of muscle DNA, also had selective loss of mtDNA-encoded subunits (patients P9 and P23 in Table 2), whereas a third patient with mtDNA depletion had patchy loss of staining of all subunits (P29).
Group 2 (Table 2)
The majority of patients did not have the same pattern of immunostaining as those with known mtDNA mutations. Thirteen patients had reduced staining of all subunits (group 2A in Table 2), whilst 10 had normal staining of all subunits despite reduced COX activity on histochemical staining (group 2B in Table 2).
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Discussion |
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TopAbstractIntroductionPatientsMaterial and methodsResultsDiscussionReferences |
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As neither clinical, biochemical nor histochemical features enable patients with mtDNA or nuclear mutations to be differentiated reliably, we sought to determine whether COX subunit expression patterns detected immunohistochemically can distinguish between mtDNA defects and nuclear defects in COX deficiency.
COX subunit expression pattern in patients with mtDNA mutations
Selective and marked reduction of mtDNA-encoded COX subunits I and II was observed in all five patients with a known mtDNA mutation. This reduction was restricted to COX-deficient fibres, all subunits appearing normal in COX-positive fibres. In addition, less marked reduction of the nuclear-encoded COX subunit VIc was also observed in COX-deficient fibres in these patients. Immunostaining with antibodies directed against nuclear-encoded subunits COX IV and Va, however, was normal in all muscle fibres from all five patients with known mtDNA mutations.
Selective loss of mitochondrial-encoded COX subunits has been observed previously in patients with mtDNA deletions (Mita et al., 1989; Moraes et al., 1992; Taanman et al., 1996), a point mutation in the mitochondrial tRNAleu(CUN) gene (Fu et al., 1996) and a 15-bp microdeletion in the COX III gene (Keightley et al., 1996). Levels of COX subunit IV were preserved in the patients studied by Mita and colleagues (Mita et al., 1989), Moraes and colleagues (Moraes et al., 1992) and Fu and colleagues (Fu et al., 1996). However no antibodies against other nuclear-encoded subunits were used in these studies and so the fate of other nuclear-encoded COX subunits was not determined in these patients. Taanman and colleagues examined immunostaining of COX Va, Vb and VIc as well as IV in a patient with chronic progressive external ophthalmoplegia harbouring a mtDNA deletion, and found greatly reduced levels of COX I, II, Va, Vb and VIc in COX-negative fibres, with relatively preserved levels of COX IV (Taanman et al., 1996). The patient with the COX III microdeletion had little or no reactivity with antibodies to COX I and VIc but intact immunostaining of subunit IV (Keightley et al., 1996). Immunoblot analyses have also demonstrated relative preservation of COX IV and Va and reduction of COX VIc in 
0 cells lacking mtDNA-encoded subunits (Taanman et al., 1996; Marusich et al., 1997).
Differential loss of immunostaining of nuclear-encoded COX subunits in patients with known mtDNA mutations may be related to the quaternary structure of the holoenzyme, which was recently determined for the bovine enzyme (Tsukihara et al., 1996). Subunit VIc forms a dumb-bell, with a transmembrane helix in contact with helix I of subunit II. It is possible that the stability of subunit VIc may be impaired when there are reduced levels of subunit II, since the two subunits are intimately related. Subunits IV and Va, on the other hand, have interactions with several nuclear subunits. These may form stable partial complexes in the absence of mitochondrial subunits.
In the present study, the COX-deficient patients who had loss of subunits I and II were not typical of the group of patients usually associated with mtDNA defects. Some of the patients presented in the neonatal period with congenital lactic acidosis, variably associated with encephalomyopathy, cardiomyopathy, liver disease and renal tubulopathy. Two had Leigh syndrome confirmed neuropathologically. In the past these patients may have been assumed to have nuclear defects. Three patients did have a ragged-red fibre myopathy presenting late in childhood, a common phenotype associated with mtDNA defects. One of these had selective loss of subunit II with normal immunostaining of subunit I. A point mutation has been identified in the COX subunit II gene in this patient, who is described in detail elsewhere (Rahman et al., 1999). Loss of COX II immunostaining in this patient was associated with reduced immunostaining of subunit VIc. This may be seen as further evidence that subunit II is required for stability of subunit of VIc. None of the 12 patients with selective loss of mtDNA-encoded COX subunits in this study has the common mtDNA point mutations at nucleotides 3243, 8344 or 8993. Southern blot analysis has revealed mtDNA depletion in two of these patients, including one with pathologically proven Leigh syndrome. Direct sequencing of mtDNA is in progress for the remaining nine patients.
COX subunit expression patterns in the mtDNA depletion syndrome
COX subunit expression has been studied previously in 10 patients with the mtDNA depletion syndrome (Moraes et al., 1991; Tritschler et al., 1992; Macmillan and Shoubridge, 1996; Taanman et al., 1997; Marusich et al., 1997). Eight had selective loss of mitochondrial-encoded subunits (I and/or II), with variable loss of nuclear subunits. Two patients, however, had homogeneous reduction of all COX subunits compared with controls. In our study, two patients with mtDNA depletion had loss of mitochondrial-encoded subunits whilst a third had loss of all subunits. We therefore confirm that different patterns of subunit expression may be seen in this syndrome. This may reflect genetic heterogeneity or, alternatively, may be related to the stage in the course of the disease (Taanman et al., 1997). A recent study of human leukaemic cells (Molt-cells) which lack mitochondrial protein synthesis suggested that all COX subunits are lost if the mitochondrial translation defect is sufficiently severe (Nijtmans et al., 1995b). Studies of 0 cells, which lack mtDNA, have yielded similar results (Taanman et al., 1996; Marusich et al., 1997).
Other COX subunit expression patterns
The remainder of our group of patients had COX subunit patterns falling into one of two broad groups: intact staining of all subunits tested, or reduced staining of all subunits. We postulate that these patients are likely to have nuclear DNA defects. To date no mutations have been described in nuclear-encoded COX subunits; therefore, patients with known mutations cannot be used for comparison. However, pedigree analysis indicates autosomal recessive inheritance in some distinct clinical entities associated with COX deficiency, such as fatal infantile myopathy and Leigh syndrome (Rahman et al., 1996). The present study suggests that Leigh syndrome associated with COX deficiency is aetiologically heterogeneous. Two patients had loss of immunostaining of mitochondrial subunits (including one with the mtDNA depletion syndrome) whilst others had loss of staining of all subunits or normal staining of all subunits. Sequence analysis of the SURF1 gene is in progress in these patients with COX-deficient Leigh syndrome.
In patients with intact immunostaining of all subunits despite reduced or absent COX staining, there seems to be reduced function despite apparently normal assembly of the holoenzyme. These patients may have a kinetic defect. A single patient with COX deficiency has been described who had an altered Km (Michaelis constant) for reduced cytochrome c (Nijtmans et al., 1995a). Studies in cultured fibroblasts from this patient revealed decreased COX activity but normal synthesis, assembly and stability of both mitochondrial and nuclear-encoded COX subunits. This patient was thought to have a defect in one of the nuclear subunits of the enzyme, as mtDNA analysis failed to identify a mutation and the parents were consanguineous. In other patients it is theoretically possible that a mtDNA defect involving a COX gene could alter the kinetic properties of the enzyme but allow normal assembly and therefore normal immunostaining of COX subunits.
Conclusions
We have identified a specific pattern of COX subunit loss in COX deficiency secondary to mtDNA mutations. We now use COX subunit immunohistochemistry to identify patients likely to have mtDNA defects and, after excluding mtDNA depletion by Southern blot analysis, selectively sequence mtDNA in these patients. The majority of children with COX deficiency do not have selective loss of mitochondrially encoded subunits. It therefore seems likely that nuclear gene defects account for a large proportion of childhood-onset COX deficiency.
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Acknowledgments |
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We wish to thank Professor P. T. Clayton and Drs J. E. Collins and R. A. H. Surtees, who allowed us to study patients in their care. Monoclonal antibodies against COX were developed in the laboratory of Dr R. A. Capaldi. S.R. is the recipient of a Medical Research Council clinical training fellowship. The study received support from the Wellcome Trust, the Research Trust for Metabolic Diseases in Children and the Muscular Dystrophy Group (UK).
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Received June 7, 1999. Revised August 27, 1999. Accepted September 28, 1999.
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 Y. Y. Li, D. Chen, S. C. Watkins, and A. M. Feldman Mitochondrial Abnormalities in Tumor Necrosis Factor-{alpha}-Induced Heart Failure Are Associated With Impaired DNA Repair Activity Circulation, November 13, 2001; 104(20): 2492 - 2497. [Abstract] [Full Text] [PDF]
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 B. J. Hanson, R. Carrozzo, F. Piemonte, A. Tessa, B. H. Robinson, and R. A. Capaldi Cytochrome c Oxidase-deficient Patients Have Distinct Subunit Assembly Profiles J. Biol. Chem., May 4, 2001; 276(19): 16296 - 16301. [Abstract] [Full Text] [PDF]
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